Team:TU Darmstadt/Protocols/Protein Expression

Standard Protocol Protein Expression Est13

Materials

• DYT medium
• 100 mL and 3 L flasks
• E.coli [http://www.neb.com/nebecomm/products/productC3026.asp BL21 SHuffle] pET26b-Est13
• 1 mM stock of isopropyl-ß-D-thiogalactopyranosid (IPTG)
• incubator
• photometer
• kanamycin
• ice

Procedure

1. Inoculation of 50 mL DYT medium in the 100 mL flask with E.coli BL21 SHuffle containing pET26b plasmid with the sequence for the protein Est13. Addition of the 15 µL kanamycin to the medium.
2. Incubation at 180 repulsion per minute (rpm) at 30°C to an OD₆₀₀= 4.
3. Transferation of the starter culture into 1 L DYT medium in a 3 L flask resulting in an OD₆₀₀= 0.2. Addition of 300 µL kanamycin.
4. Incubation to an OD₆₀₀= 0.6 at 180 rpm and 30°C.
5. Incubation for 15 min. on ice.
6. Induction of the proteinexpression with 0.8 mL of IPTG (stock conentration 1mM).
7. Incubation of the cell suspension over night at 180 rpm at 16°C.